Abstract LB-148: Characterization of an off-target RNAi genomic screen hit identifies GPRC6A as a novel suppressor of metastatic chemotaxis and invasiveness

Abstract

Abstract Metastatic events define the lethal phenotype of prostate cancer; however, as a result of enhanced and more widespread screening, many cancers are now detected prior to the presentation of overt metastases. In these cases, there is a need to characterize the molecular and genetic profile of primary tumors to use as accurate predictors of aggressive, metastatic disease. The following study utilizes unconventional analysis of off-target data from two genome-wide RNAi screens for metastasis suppressor genes in prostate and breast cancer models to identify novel markers and potential therapeutic targets of advancing disease. One screen enriched for increased Matrigel invasiveness by human T-47D breast cancer clones whereas the other screen selected for increased macrometastasis formation in distant organs after orthotopic xenograft of human LNCaP prostate cancer clones. A particular hairpin targeting apolipoprotein L-II (APOL2) was enriched in both screens and this shRNA sequence increases LNCaP invasion and chemotaxis in transwell assays. Notably, knockdown of APOL2 by alternative RNAi sequences does not induce LNCaP invasion indicating that off-target effects of the specific shRNA (called si/shAPOL2*) are responsible for the phenotype. Microarray profiling identified 132 transcripts differentially downregulated by siAPOL2* and sequence analysis reveals similarity to a number of microRNA (miR) seed sequences, miR-8087, miR-548u, miR-4432, and miR-543. With the exception of miR-548u, transfection with the other miR mimics recapitulates the enhanced chemotactic phenotype induced by siAPOL2* in LNCaP cells. Both miR-4432 and miR-543 expression varies in a panel of prostate cancer cell lines (LNCaP, C4-2, PC3, and DU-145), whereas miR-8087 is not detected by qPCR in these cell lines and has no associated expressed sequence tags (ESTs) in the GenBank database. Amplifications of miR-4432 and miR-543 are present in large numbers of patients across multiple cancer types in The Cancer Genome Atlas (TCGA) datasets. Microarray data and qPCR validation suggests G-protein coupled receptor family C group 6 member A (GPRC6A) as a candidate off-target transcript affected by siAPOL2*. Indeed, GPRC6A was also identified independently in the LNCaP orthotopic xenograft RNAi screen, and independent siRNA-mediated knockdown of GPRC6A in LNCaP cells increases chemotaxis. Further investigation is underway to determine whether miR-4432 or miR-543 mediate their effects through the regulation of GPRC6A, presumably through 3’UTR miRNA binding sites. This study demonstrates that the analysis of off-target effects in RNAi screens is a viable method to identify functionally relevant microRNAs contributing to regulation of chemotaxis in metastatic disease. Citation Format: Henry G. Withers, Irwin H. Gelman. Characterization of an off-target RNAi genomic screen hit identifies GPRC6A as a novel suppressor of metastatic chemotaxis and invasiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-148. doi:10.1158/1538-7445.AM2017-LB-148